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. 2026 Feb 1;37:102867. doi: 10.1016/j.mtbio.2026.102867

Identification of a myosin 1B-binding aptamer for fluorescence imaging and targeted therapy of esophageal squamous cell carcinoma

Zhaoting Wang a,1, Xiaoxiong Xiao b,c,1, Tianlu Zhang a,1, Xiao Li a, Mengmeng Ji a, Yongqi Qian a, Xue Bai a, Xin Li a,d, Jing Lu a,d, Jinlu Tang a, Kangdong Liu a,d,e, Zhaohui Li f,⁎, Baoyin Yuan a,d,⁎⁎
PMCID: PMC12890841  PMID: 41685366

Abstract

Esophageal squamous cell carcinoma (ESCC), a prevalent subtype of esophageal cancer, poses a significant global health challenge. The current diagnostic and therapeutic approaches for ESCC are inadequate, highlighting the urgent need for the development of novel recognition molecules and the identification of new therapeutic targets to facilitate early diagnosis and targeted therapy. In this work, several aptamers with high affinity to target KYSE30 cells are screened through Cell-based Systematic Evolution of Ligands by EXponential enrichment (Cell-SELEX). These aptamers show distinct binding to multiple cancer cells including ESCC, gastric cancer and liver cancer cells. Meanwhile, a truncated aptamer Z4-6 that retains comparable binding affinity is achieved via sequence optimization. With Z2 and Z4-6 aptamers as the recognition elements, Cy5-labeled fluorescent aptamer probes have demonstrated the ability to specifically recognize ESCC tissues while sparing adjacent non-cancerous tissues, suggesting their potential utility as detection probes in the clinical diagnosis of ESCC. Furthermore, myosin 1B has been identified as the molecular target of the aptamer Z4-6 through pull-down and RNA interference assays, underscoring its promise as a tumor biomarker and therapeutic target. The Z4-6 aptamer has been employed to construct Z4-6-Dox conjugates via noncovalent loading with doxorubicin (Dox) for the targeted therapy of ESCC. In vitro cytotoxicity assays have revealed that Z4-6-Dox selectively induces cytotoxicity in KYSE30 cells. Notably, the Z4-6 aptamer demonstrates in vivo tumor-targeting capabilities, and Z4-6-Dox effectively inhibits tumor cell growth with reduced cardiotoxicity. This study contributes valuable molecular recognition tools and identifies a potential target for the precise diagnosis and targeted therapy of ESCC.

Graphical abstract

A myosin 1B-binding aptamer against ESCC is identified through Cell-SELEX, and it can be used for fluorescence imaging and targeted therapy of ESCC.

Image 1

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1. Introduction

Esophageal cancer is a prevalent malignant tumor that poses a significant threat to human health, ranking seventh in mortality and eleventh in incidence globally [1]. This cancer primarily consists of two subtypes: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) [2]. Notably, over 70% of esophageal cancer cases are reported in China, with ESCC making up about 90% of these diagnoses [3]. The early symptoms of ESCC are often subtle, leading to a majority of patients being diagnosed at an advanced stage with metastasis, which contributes to a high recurrence rate and poor prognosis. The five-year overall survival rate for these patients is less than 20% [4]. Therefore, early diagnosis and treatment of ESCC are crucial. Currently, the main treatment methods for ESCC in clinical practice include traditional surgery, chemotherapy, and radiotherapy [5]. Chemotherapy remains the first-line treatment for ESCC [6]; however, it lacks specificity in targeting tumor cells, resulting in significant side effects and limiting its therapeutic efficacy [7]. Targeted chemotherapy utilizes recognition molecules to selectively deliver chemotherapeutic agents to tumor sites, thereby reducing side effects and enhancing treatment efficacy. In recent years, targeted therapy for esophageal cancer has garnered significant attention. Although therapeutic targets such as EGFR, VEGFR, and HER2 have been employed in the treatment of ESCC, their effectiveness is substantially limited [8]. Consequently, there is an urgent need to develop novel targeting recognition molecules and discover novel therapeutic targets for the early diagnosis and targeted therapy of ESCC.

Aptamers are short single-stranded DNA or RNA sequences that screened through the Systematic Evolution of Ligands by EXponential enrichment (SELEX) [9]. These aptamers exhibit high affinity and specificity for their targets, and offer several advantages over protein antibodies, including ease of synthesis and modification, low cost, flexible design, and low immunogenicity [10]. Aptamers targeting tumor cells are screened through Cell-SELEX, which employs tumor cells as targets for aptamer selection [[11], [12], [13]]. As novel tumor-specific recognition molecules, aptamers are widely used in cancer research [[14], [15], [16], [17]]. When conjugated with signaling reporters such as fluorescent groups and radioisotopes, aptamers serve as effective detection probes for the molecular diagnosis of cancers [[18], [19], [20], [21]]. Owing to their exceptional tumor-targeting capabilities, aptamers are employed as specific recognition elements for targeted drug delivery [[22], [23], [24]]. Notably, the molecular targets of aptamers that screened through Cell-SELEX remain unknown. Utilizing tumor cell-specific aptamers as the fishing baits may facilitate the discovery of novel targets for targeted cancer therapy [[25], [26], [27]].

Recently, several aptamers have been reported to detect and treat ESCC. In our previous studies, with poorly differentiated ESCC cells as target cells and highly differentiated ESCC cells as control cells, aptamers capable of binding to ESCC cells were developed through Cell-SELEX [28,29]. Specifically, the A2 aptamer, selected against ESCC KYSE410 cells, enables fluorescent imaging and targeted chemotherapy of ESCC, with integrin beta 1 identified as the molecular target [28]. The SYL-6 aptamer, which binds to various cancers including ESCC, has been verified to target prohibitin 2 [30]. However, these aptamers also exhibit non-negligible binding to normal esophageal epithelial cells, which is a concern. The EA1 and EA2 aptamers have been reported to specifically bind to ESCC KYSE150 cells, yet their molecular targets remain unidentified, potentially limiting their applications [31].

In this work, to achieve an aptamer targeting to ESCC with high specificity and specific molecular target, ESCC KYSE30 cells were used as target cells for positive selection and normal esophageal epithelial Het-1A cells were used as control cells for negative selection through Cell-SELEX (Scheme 1). After ten rounds of selection, we identified three aptamers exhibiting high binding specificity and affinity for KYSE30 cells. Additionally, through sequence optimization, we obtained a truncated aptamer, Z4-6, which maintained comparable binding affinity. When labeled with the Cy5 fluorescent dye, the Z2 and Z4-6 aptamers enabled specific fluorescence imaging of ESCC tissues. Furthermore, myosin 1B was validated as the molecular target of Z4-6, highlighting its potential for targeted therapies of ESCC. Due to its high GC content, Z4-6 was further used as a carrier to deliver Doxorubicin (forming Z4-6-Dox) for targeted therapy of ESCC. In vitro cytotoxicity assays demonstrated that Z4-6-Dox selectively induced cytotoxicity in KYSE30 cells. In vivo experiments corroborated that Z4-6 effectively targets tumor sites, and Z4-6-Dox inhibits tumor cell growth while reducing cardiotoxicity. This work presents valuable molecular recognition tools and a promising target for precise diagnosis and targeted therapy of ESCC.

Scheme 1.

Scheme 1

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Schematic representation of the Cell-SELEX process. ESCC KYSE30 cells were used as target cells for positive selection and normal esophageal epithelial Het-1A cells were used as control cells for negative selection.

2. Experimental sections

2.1. Primary DNA library and PCR primers

The primary DNA library (5′-AGAAGGAAGGAGAGCGACACNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNTATCAGTGGTCGGTCGTCAT-3′) comprises a central random region of 40 nucleotides flanked by two constant primer regions, each consisting of 20 nucleotides. The forward primer (5′-FAM-AGAAGGAAGGAGAGCGACAC-3′) was labeled with FAM dye to monitor the selected libraries, while the reverse primer (5′-Biotin-ATGACGACCGACCACTGATA-3′) was labeled with biotin to enable the preparation of single-stranded sequences.

2.2. Cell-SELEX process

50 μL primary DNA library (100 μM, in PBS) was first mixed with 50 μL binding buffer (0.01M PBS containing 5 mM MgCl2·6H2O, 5 g/L glucose, 0.1 mg/mL yeast tRNA and 1 mg/mL BSA), and then heated at 95 °C for 5 min and cooled at 4 °C for 10 min. After the DNA library was diluted with binding buffer to 5 μM, target KYSE30 cells were blocked with random sequences and then incubated with the prepared library at 4 °C. The cells were collected and heated at 95 °C for 10 min after washing with washing buffer (0.01M PBS containing 5 mM MgCl2·6H2O and 5 g/L glucose). Following centrifugation, the supernatant was collected to serve as the template for PCR. 100 μL streptavidin-coated sepharose beads were mixed and incubated with the PCR products for 30 min. Subsequently, the mixture was treated with NaOH solution (200 mM) for 15 min after washing. The mixture was centrifuged, and the supernatant containing the single-stranded library was retained. After desalted by NAP-5 column, the library was quantified and dried. Negative selection was conducted after the third round of selection. Control Het-1A cells were incubated with the library at 4 °C, and the supernatant was collected for positive selection. During the selection process, the incubation time of the target cells with the library was reduced from 1 h to 40 min, while the incubation time of the control cells with the library was risen from 30 to 40 min. The saturated library was subsequently subjected to cloning and sequencing by Sangon Biotechnology.

2.3. Flow cytometry analysis

Usually, 1 × 105 target KYSE30 or control Het-1A cells were incubated with 5 μL FAM-labeled aptamers (10 μM) in 200 μL binding buffer at 4 °C for 40 min. The cells were washed and resuspended in 400 μL binding buffer for flow cytometry analysis (BD Biosciences, USA). To determine the dissociation constants (Kd) of the aptamers, KYSE30 cells were incubated with 0, 5, 10, 25, 50, 100, 250, and 500 nM FAM-labeled aptamers, respectively. The Kd was calculated using the one-site saturation equation, Y = Bmax X/(Kd + X), where X represents the aptamer concentration and Y denotes the fluorescence intensity. To examine the binding temperature, KYSE30 cells were incubated with the FAM-labeled aptamers at 4, 25, 37 and 40 °C, respectively. To examine the target type, KYSE30 cells were initially treated with trypsin for 4 min or Proteinase K for 3 and 10 min, followed by incubation with the FAM-labeled aptamers. To examine the binding in different buffers, KYSE30 cells were incubated with the FAM-labeled aptamers in PBS, FBS, RPMI-1640, and RPMI-1640 supplemented with 10% FBS.

2.4. Confocal imaging

KYSE30 and Het-1A cells (approximately 5 × 104 cells) were seeded into confocal dishes and cultured for 24 h. Subsequently, the cells were incubated with FAM-labeled aptamers (250 nM) at 4 °C for 40 min. Following incubation, unbound aptamers were removed through washing, and the cells were imaged by a laser scanning confocal microscope (Olympus, Japan). To examine the internalization of the aptamer-Dox, KYSE30 and Het-1A cells were treated with 4 μM aptamer-Dox at 37 °C for 2.5 h in a medium without FBS. After washing, the cells were stained with Hoechst33342 dye for 10 min prior to confocal imaging. For tissue imaging with aptamer probes, ESCC tissue chips were dewaxed in xylene and dehydrated through a gradient alcohol series (75–100%). After treatment with a boiled alkaline repair solution, the tissue chips were washed with PBS and preincubated with random DNA sequences (250 nM) at 4 °C for 30 min. Thereafter, the tissue chips were stained with Cy5-labeled aptamer probes at 4 °C for 40 min and subjected to confocal imaging following a washing step.

2.5. Mass spectrometry analysis

KYSE30 cells were lysed by hypotonic buffer (10 mM Tris-HCl, 0.1 mM PMSF, 1 × cocktail protease inhibitors), and the membrane proteins were extracted using 1% Triton X-100 in hypotonic buffer for subsequent aptamer-based pull-down assays. 100 μL of Sepharose beads were blocked with 5% BSA, while the membrane protein sample was blocked with 0.5 mg/mL salmon sperm DNA at 4 °C for 1 h. The beads were initially incubated with the protein sample at 4 °C for 1 h. The resulting supernatant was then incubated with biotin-labeled random DNA (250 nM) at 4 °C for 1 h, followed by incubation with an additional 100 μL of beads. After centrifugation, the supernatant was collected to incubate with biotin-labeled aptamer (250 nM) at 4 °C for 1 h and subsequently with another 100 μL of beads. The protein-bound beads underwent washing and were resuspended in loading buffer. After boiling for 10 min, the supernatants were analyzed by SDS-PAGE, and the gel was stained by Coomassie blue. Differential protein bands were excised and analyzed by LC−MS/MS by Genechem. (Shanghai, China).

2.6. Western blot

KYSE30 and Het-1A cells were lysed using RIPA buffer, and the supernatant was collected. Protein concentration was quantified using a BCA kit. The protein samples were first analyzed by SDS-PAGE, followed by transfer to a polyvinylidene difluoride membrane. After blocked with 5% skim milk, the membrane was incubated with an anti-MYO1B primary antibody at 4 °C overnight. Thereafter, an HRP-labeled secondary antibody was added and incubated at 25 °C for 2 h. Protein visualization was achieved using an ultrasensitive ECL luminescent solution, and imaging was conducted with Image Quant (GE Healthcare, USA).

2.7. RNA interference

The siRNA sequences of MYO1B (siRNA-1 sense, GUGUGGUUAUAUCUGUUAATT; siRNA-2 sense, AGAAGUUAAUCAAGUUAAATT; and siRNA-3 sense, GGAGAAAGUUUCAACUACATT) and a negative control siRNA sequence (sense, UUCUCCGAACGUGUCACGUTT) were synthesized by Sangon Biotechnology (Shanghai, China). KYSE30 cells were transfected with siRNAs for 48 h using Lipofectamine 3000 (Thermo Fisher, USA). Protein extracts from these cells were detected by western blot with an anti-MYO1B antibody. KYSE30 cells transfected with siRNA sequences were incubated with FAM-labeled Z4-6 for binding assays by flow cytometry.

2.8. Preparation and characterization of the aptamer-Dox complex

Because Dox can be embedded into GC base pairs through non-covalent interactions, nucleic acids are ideal as carriers for Dox delivery [32,33]. To prepare the aptamer-Dox complex, 10 μM aptamer was mixed with 50 μM Dox in PBS at room temperature for 30 min. To verify Dox loading with aptamers, the aptamer-Dox complex was analyzed using mass spectrometry by Sangon Biotechnology. To investigate the Dox loading capacity, Dox was fixed at 2 μM, and various aptamer-to-Dox ratios (0, 1:20, 1:15, 1:10, 1:7.5, 1:5, 1:3, and 1:2) were prepared as above mentioned, and Dox fluorescence was examined by F-7000 fluorescence spectrometer (Hitachi, Japan).

2.9. CCK8 analysis

KYSE30 cells (5000 cells/well) and Het-1A (6000 cells/well) cells were seeded into 96-well plates and cultured until they adhered. Subsequently, the cells were incubated with Dox (4 μM), an aptamer (0.8 μM), and an aptamer-Dox complex (0.8 μM) in RPMI-1640 medium for 2.5 h. After washing, a complete medium was added for a further 48-h culture period. Subsequently, 10 μL of CCK8 solution was added into each well and incubated for 2 h, after which the absorbance at 450 nm was recorded by a microplate reader.

2.10. Calcein AM/PI staining

KYSE30 and Het-1A cells (6 × 104 cells/well) were seeded into 6-well plates and cultured until adherence. Subsequently, the cells were incubated with Dox (4 μM), an aptamer (0.8 μM), and an aptamer-Dox complex (0.8 μM) in RPMI-1640 medium for 2.5 h. After washing, a complete medium was added for a further 48-h culture period. The cells were then detached by trypsin, collected, and stained with 0.5 mL of Calcein AM/PI solution at 37 °C for 30 min. Finally, the stained cells were transferred onto glass slides and imaged by a fluorescence microscope.

2.11. Xenograft mouse model

BALB/c nude mice (male, 6 weeks old) were provided by SPF Biotech (Beijing, China). To construct a xenograft mouse model, approximately 1 × 107 KYSE30 cells were subcutaneously injected into the dorsal region of the mice. Upon reaching a tumor volume of approximately 200 mm3, the mice were subjected to in vivo experiments. The animal protocols were approved by the Medical Ethics Committee of Zhengzhou University.

2.12. In vivo experiments

For in vivo fluorescence imaging, 2.5 nmol of Cy5-labeled Random sequences or aptamer probes were injected into the mice through tail vein. Fluorescence signals were recorded in real time by the IVIS Lumina II Imaging System (Perkin Elmer, USA). Additionally, ex vivo imaging was performed on isolated tumors. For in vivo antitumor treatment studies, mice were separated into four groups (n = 5): 1) PBS; 2) Z4-6 aptamer, 3) Free Dox, 4) Z4-6-Dox complex. The dosage of Dox was set at 2 mg/kg, with a molar ratio of Z4-6 to Dox of 1:5. Drug administration was conducted every two days through tail vein injection, for a total of five administrations. Tumor size and body weight were recorded every two days. Tumor volume was calculated using the formula: tumor volume (mm3) = (length × width2)/2. The mice were sacrificed at 11 days, and the weight of the striped tumors were measured. The blood of treated mice was collected for blood biochemistry analysis. The tissues of major organs from treated mice were subjected to hematoxylin and eosin (HE) staining. Heart tissues were additionally subjected to TUNEL and Masson staining.

2.13. Data analysis

Data analysis was conducted using Graphpad Prism 8.0 and SPSS 22.0 software. Results were expressed as the mean ± standard deviation (SD). An independent t-test was employed to assess differences between two groups, while one-way analysis of variance (ANOVA) was used for comparisons among multiple groups. P < 0.05 was considered statistically significant.

3. Results and discussion

3.1. Aptamers that bind to ESCC KYSE30 cells were selected through Cell-SELEX

To screen DNA aptamers targeting to ESCC, Cell-SELEX was performed by using ESCC KYSE30 cells as target cells for positive selection and normal esophageal epithelial Het-1A cells as control cells for negative selection. The selection process was consistent with our previous methods with minor modifications [28,34,35]. After ten rounds of selection, the binding ability of the selected libraries was analyzed by flow cytometry. As shown in Fig. 1A, the fluorescence intensity of KYSE30 target cells incubated with the ssDNA libraries significantly enhanced with increasing selection rounds, while Het-1A control cells just showed weak binding to these libraries, indicating that the ssDNA library specific to target cells was effectively enriched during the selection process. Given that the ninth round of the ssDNA library almost reached the highest binding to the target cells, it was used for cloning and sequencing. A total of 155,061 DNA sequences were sequenced, and the top 23 sequences, each with a copy number exceeding 1,000, were subjected to homology and phylogenetic tree analysis. The results showed a high degree of homology among the selected 23 sequences (Fig. S1), which were categorized into several families (Fig. S2). Based on high copy number and distant homology as evaluation criteria, sequences 1–6 were picked out and designated as Z1-6 aptamer candidates for subsequent experiments.

Fig. 1.

Fig. 1

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Selection and identification of DNA aptamers specific to ESCC. (A) Flow cytometry assays of the binding of the selected libraries to KYSE30 and Het-1A cells. (B) Flow cytometry assays of the binding of Z1-6 aptamer candidates to KYSE30 and Het-1A cells. (C) The binding signal-to-background ratio (SBR) calculation in Fig. 1B. Fluorescence intensity of random ssDNA was used as the background signal. (D) Confocal imaging of KYSE30 cells incubated with FAM-labeled Z1, Z2, and Z4 aptamers. The scale bar is 50 μm. (E) Determination of dissociation constant (Kd) of Z1, Z2, and Z4 aptamers for KYSE30 cells by flow cytometry. FAM-labeled random ssDNA was used as the negative control.

The binding ability of Z1-6 to KYSE30 target cells and Het-1A control cells was detected by flow cytometry. As shown in Fig. 1B, aptamers Z1, Z2, and Z4 displayed strong binding to KYSE30 target cells, while demonstrating weak binding to Het-1A control cells. This trend was corroborated by the quantitative analysis of the binding signal-to-background ratio (SBR) (Fig. 1C). Consequently, Z1, Z2, and Z4 were selected as the aptamers of interest for further investigation. Confocal imaging was then used to verify the specific binding of these aptamers. As shown in Fig. 1D, bright fluorescence was observed on KYSE30 target cells stained with FAM-labeled aptamers, particularly with the Z4 aptamer. In contrast, no obvious fluorescence signals were observed on Het-1A control cells after aptamer staining (Fig. S3). These results further verified specific binding of Z1, Z2, and Z4 aptamers to the target cells. Subsequently, the dissociation constants (Kd) of Z1, Z2, and Z4 aptamers for KYSE30 target cells were determined by flow cytometry. As demonstrated in Fig. 1E, the Kd values for Z1, Z2, and Z4 were in the low nanomolar range (Z1: 6.72 ± 1.05 nM, Z2: 15.72 ± 5.82 nM, Z4: 35.97 ± 5.86 nM), indicating a high binding affinity of these aptamers to the KYSE30 cells. In addition, the binding of Z1, Z2, and Z4 to target cells under different temperature was analyzed. The results showed that Z1, Z2, and Z4 can bind to target cells at 4, 25, 37, and 40 °C (Fig. S4), indicated that these aptamers may be feasible for in vitro diagnosis and in vivo applications.

3.2. Z1, Z2, and Z4 aptamers bind to multiple cancer cells

To evaluate the binding specificity of Z1, Z2, and Z4 aptamers, thirteen cancer cell lines alongside two normal cell lines were used to detect the binding of aptamers by flow cytometry. As shown in Fig. 2, Z1 and Z2 exhibited strong binding to ESCC KYSE30 cells and gastric cancer HGC-27 cells. In contrast, Z4 demonstrated high binding signals with ESCC KYSE30 and KYSE70 cells, gastric cancer HGC-27 cells, and liver cancer HepG2 cells, while displaying low binding signals with ESCC KYSE450, KYSE150, KYSE410, ECa109 cells, liver cancer Huh-7 cells, breast cancer MCF-7 cells, renal cancer Renca cells, lung cancer EBC-1 cells, colorectal cancer DLD-1 cells, normal renal epithelial 293T cells, and normal esophageal epithelial Het-1A cells. The calculation of binding SBR further corroborated these findings (Fig. S5). Notably, these aptamers differ from previously reported ESCC-binding aptamers due to their distinct binding profiles across various cell types [28,29,31]. Additionally, Z1 and Z2 had similar binding specificity and signal intensity, suggested that their binding targets may be the same molecules. Consequently, Z2 was chosen for further evaluation in subsequent experiments. The ability of Z2 and Z4 to bind to a variety of cancer cells can be attributed to the high expression levels of their targets in these cancer cells, including those of gastric cancer and liver cancer cells. It was worth noting that Z2 and Z4 selectively recognize only certain ESCC cells, suggesting high heterogeneity among ESCC cells. Given their distinct recognizing ability to multiple cancer cells, Z2 and Z4 aptamers hold great potential in cancer diagnosis and targeted therapy.

Fig. 2.

Fig. 2

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Flow cytometry assays of the binding of Z1 (blue), Z2 (orange), and Z4 (green) aptamers to different cell lines. Random ssDNA (red) was used as the control probe. Aptamers and random ssDNA were labeled with FAM group for collecting fluorescence signals.

3.3. Truncated aptamers are obtained through sequence optimization

Primary aptamers selected through Cell-SELEX typically possess relatively long sequences, encompassing primer regions and redundant bases, which may adversely impact their binding to targets [36,37]. Sequence optimization of these aptamers not only reduces the costs of aptamer synthesis, but also improves their binding ability. The Z2 aptamer was first subjected to sequence optimization. The secondary structure of Z2 was simulated by NUPACK software. By preserving the main stem-loop and cutting the primer regions (Fig. S6A), truncated aptamers Z2-1 and Z2-2 were obtained. The results of flow cytometry assays showed that the binding of Z2-1 decreased dramatically, while Z2-2 showed only a slight decrease (Fig. S6B), indicated that the primer regions are crucial for Z2 binding, and the terminal stem-loop structure plays a pivotal role in its binding. Considering better binding of intact Z2, it was still used in next evaluation. The Z4 aptamer was then subjected to sequence optimization. As shown in Fig. 3A, Z4 displayed an overall three-way structure, comprising two stem-loops and one stem structure. The optimization strategies are displayed in Fig. 3A, Z4-1: retain the three-way structure, Z4-2: cut primer regions, Z4-3: retain loop 1 and 2, Z4-4: retain loop 1, Z4-5: retain loop 2, Z4-6: cut the terminal redundant bases. Flow cytometry was used to analyze the binding of Z4-1 to Z4-6 truncated aptamers to KYSE30 target cells. The results showed that Z4-2 to Z4-5 lost their binding to target cells, and the binding of Z4-1 was decreased. In contrast, Z4-6 maintained a great binding ability to the target cells (Fig. 3B). The Kd of Z4-6 truncated aptamer for KYSE30 cells was determined to be 34.75 ± 6.45 nM (Fig. 3C), which is comparable to the intact Z4 aptamer, suggesting a high affinity of Z4-6 for the target cells. These results indicated that the three-way structure of Z4 is crucial for target cell binding. Due to its short length and strong binding ability, Z4-6 was chosen as the optimal aptamer for subsequent experiments.

Fig. 3.

Fig. 3

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Optimization and characterization of Z4 aptamer. (A) Optimization strategies of Z4 aptamer. The secondary structures of aptamers were simulated by NUPACK software. (B) Flow cytometry assays of KYSE30 cells incubated with FAM-labeled Z4-1 to Z4-6 truncated aptamers. Corresponding binding SBR was calculated. FAM-labeled random DNA was used as the control probe. (C) Determination of Kd of Z4-6 truncated aptamer for KYSE30 target cells. (D) Agarose gel electrophoresis assays of Z2 and Z4-6 aptamers (3 μM) in 10% FBS for different durations. The relative gray value was calculated by ImageJ software. (E) CCK8 assays of KYSE30 and Het-1A cells treated with different concentrations of Z2 and Z4-6 aptamers. Random ssDNA was used as the control. The asterisks indicate significance, ∗∗P < 0.01, ∗∗∗P < 0.001.

The stability of aptamers under complex conditions is a critical determinant of their efficacy in biomedical applications. To evaluate the stability of the aptamers, Z2 and Z4-6 were incubated in 10% FBS for different time and analyzed by agarose gel electrophoresis. The results showed that both Z2 and Z4-6 can even exist for 24 h (Fig. 3D), indicating their relatively strong stability in complex conditions. Given that the Cell-SELEX process was conducted in binding buffer, we next evaluated the binding ability of aptamers in different buffers. As shown in Fig. S7, Z2 and Z4-6 maintained strong binding ability across various buffers, including PBS, FBS, and 1640 medium, suggesting their robust binding in various conditions. These results demonstrated that Z2 and Z4-6 can bind to target cells in complex conditions while retaining substantial stability. Subsequently, we focused on the effects of aptamer binding on cell viability using CCK8 assays. As shown in Fig. 3E, Z2 and Z4-6 exhibited a significant cytotoxic effect on KYSE30 target cells at a concentration of 5 μM, whereas no significant cytotoxic effects were observed on Het-1A control cells at any concentrations. We speculated that the mechanism of the killing effects is that aptamer binding may inhibit the biological function of the binding targets of aptamers, and downregulate some signaling pathways that promote cell growth and migration. These results suggested that Z2 and Z4-6 can selectively kill target cells, which is promising in drug development for targeted cancer therapy.

3.4. ESCC tissues are specifically imaged by Cy5-labeled aptamer probes

Given that the selected aptamers can bind to ESCC cell lines, we next evaluated the recognition ability of the aptamers to clinical ESCC tissues. A total of fifteen paired human ESCC and adjacent non-cancerous tissues were subjected to staining with Cy5-labeled Z2 and Z4-6 aptamer probes. As shown in Fig. 4A, bright fluorescence signals were observed in ESCC tissues stained with Z2 and Z4-6 probes, while the adjacent tissues stained with the same probes, as well as ESCC and adjacent tissues stained with random control probe, showed weak fluorescence signals. Quantitative results also showed that the fluorescence intensity of ESCC tissues stained with Z2 and Z4-6 probes were significantly higher than that of adjacent tissues stained with the aptamer probes and ESCC tissues stained with the control probe (Fig. 4B–and C). These findings suggest that Z2 and Z4-6 aptamers possess a high specificity for recognizing ESCC tissues, highlighting their potential as molecular probes for the clinical diagnosis of ESCC.

Fig. 4.

Fig. 4

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Fluorescence imaging of ESCC tissues with Cy5-labeled aptamer probes. (A) Representative images of ESCC and adjacent tissues stained with Cy5-labeled Z2 and Z4-6 aptamer probes. Cy5-labeled random ssDNA was used as the control probe. Scale bar is 50 μm. (B) Quantitative fluorescence intensity of ESCC and adjacent tissues stained with Cy5-labeled Z2 probes. (C) Quantitative fluorescence intensity of ESCC and adjacent tissues stained with Cy5-labeled Z4-6 probes. The asterisks indicate significance, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

3.5. MYO1B is identified as the molecular target of Z4-6 aptamer

In order to discover novel ESCC biomarkers, we subsequently aimed to identify the molecular target of Z4-6 aptamer. Firstly, the target type of Z4-6 was analyzed by flow cytometry. As shown in Fig. 5A, Z4-6 almost lost the binding to KYSE30 cells following treatment with protease K, suggesting that the target type of Z4-6 is likely a protein, although trypsin treatment had no effects on Z4-6 binding. Next, the total membrane proteins from KYSE30 cells were collected for Z4-6-based pull-down assays, and the captured proteins were analyzed by SDS-PAGE (Fig. 5B). A specific band around 130 kDa was collected and analyzed by LC−MS/MS. The top 20 protein candidates identified through mass spectrometry are ranked in Table S2 based on their scores. Myosin 1B (MYO1B, 131.9 kDa), which received a high score and is located on membrane, was chosen for further target identification.

Fig. 5.

Fig. 5

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Identification of molecular target of the Z4-6 aptamer. (A) Flow cytometry assays of the binding of Z4-6 to KYSE30 cells treated with trypsin and protease K. (B) SDS-PAGE assays of proteins captured by Z4-6. Lane 1 and 6, protein markers; lane 2, total membrane proteins; lane 3, beads; lane 4, random ssDNA captured proteins; lane 5, Z4-6 captured proteins. The box indicates the specific band. (C) Western blot assays of total proteins from Het-1A and KYSE30 cells with an anti-MYO1B antibody. The asterisk indicates significance, ∗P < 0.05. (D) Pull-down assays by Z4-6. The captured proteins were analyzed by SDS-PAGE, and then detected by western blot with an anti-MYO1B antibody. (E) Western blot assays of knockdown of MYO1B in KYSE30 cells using siRNAs with an anti-MYO1B antibody. NC indicates siRNA negative control. (F) Flow cytometry assays of the binding of Z4-6 to KYSE30 cells treated with the siRNAs.

The gene expression assays from GEPIA database show that MYO1B is highly expressed in esophageal carcinoma (ESCA), and other cancers including cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), colon adenocarcinoma (COAD), lymphoid neoplasm diffuse large B-cell lymphoma (DLBC), head and neck squamous cell carcinoma (HNSC), rectum adenocarcinoma (READ), skin cutaneous melanoma (SKCM), stomach adenocarcinoma (STAD), testicular germ cell tumors (TGCT), and thymoma (THYM) (Fig. S8). The wide expression of MYO1B will potentially expand the application of Z4-6. It was reported that MYO1B was correlated with tumor stage, TNM stage, and poor outcomes of ESCC. Overexpression of MYO1B can promote cell proliferation and metastasis through activating the SNAI2/cyclin D1 pathway, and thus driving tumorigenesis in ESCC [38]. These reports may explain why Z4-6 binding causes cytotoxicity (Fig. 3E). However, for underlying the mechanism of Z2 cytotoxicity, the binding target of Z2 need to be identified and the biological functions of the target need to be validated in the future. The expression levels of MYO1B in the target KYSE30 cells and control Het-1A cells were detected by western blot with an anti-MYO1B antibody. The results showed that the level of MYO1B in KYSE30 cells was significantly higher than that in Het-1A cells (Fig. 5C), corroborating the specific binding of Z4-6 to the target KYSE30 cells. Z4-6-based pull-down assays were conducted and analyzed by western blot. As shown in Fig. 5D, the proteins captured by Z4-6 specifically interacted with the anti-MYO1B antibody, suggesting that MYO1B serves as the binding target of Z4-6. To further investigate this interaction, siRNAs were used to knock down MYO1B levels in KYSE30 cells (Fig. 5E). The cells with reduced MYO1B levels were then stained with FAM-labeled Z4-6 for flow cytometry assays. As shown in Fig. 5F, the binding of Z4-6 to siRNA-treated KYSE30 cells was markedly diminished compared to cells treated with a negative siRNA control. This observation implies that the downregulation of MYO1B levels lead to reduced binding of Z4-6 to the target cells, thereby further validating MYO1B as the molecular target of the Z4-6 aptamer. Additionally, binding competition assays were performed using the Z4-6 aptamer and the anti-MYO1B antibody. However, no binding competition was found between the Z4-6 aptamer and the MYO1B antibody (Fig. S9). We speculate that their different binding sites on MYO1B contributes to this result.

3.6. Z4-6-Dox selectively kills ESCC KYSE30 cells in vitro

Doxorubicin (Dox) is a commonly used drug for cancer chemotherapy [39]. Because Dox can be simply embedded into GC base pairs through non-covalent interactions, nucleic acids are frequently employed as carriers for Dox delivery [40]. Z4-6 aptamer is rich in GC base pairs, may facilitate the targeted delivery of Dox in cancer therapy (Fig. 6A). Free Dox has intrinsic fluorescence, but this fluorescence is quenched when Dox is embedded into GC base pairs. To verify the construction of the Z4-6-Dox complex, Dox was fixed at 2 μM, as shown in Fig. 6B, Dox fluorescence gradually decreased with an increasing ratio of Z4-6 to Dox. When the ratio was 1:5, Dox fluorescence was almost the lowest, indicating that Z4-6 could load five Dox molecules. Mass spectrometry analysis was further used to verify the formation of Z4-6-Dox. As shown in Fig. S10, the Z4-6-Dox complex exhibited a greater molecular weight than Z4-6 alone, further verifying the successful construction of the Z4-6-Dox complex. Flow cytometry was then used to evaluate the binding of Cy5-labeled Z4-6-Dox to target KYSE30 cells. The results showed that Z4-6-Dox maintained a strong binding ability to the target cells (Fig. 6C), suggesting that Dox loading did not adversely affect Z4-6 binding, and that Z4-6-Dox could be utilized for targeted drug delivery.

Fig. 6.

Fig. 6

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Selective cytotoxicity of Z4-6-Dox. (A) Schematic illustration of construction of Z4-6-Dox. (B) Fluorescence spectrum of Dox incubated with various concentration of Z4-6. Dox was fixed at 2 μM. (C) Flow cytometry assays of binding of Cy5-labeled Z4-6 and Z4-6-Dox to KYSE30 cells. Confocal imaging of KYSE30 (D) and Het-1A (E) cells treated with 4 μM Dox and 0.8 μM Z4-6-Dox. Cell nuclei were stained with Hoechst33342. CCK-8 assays of KYSE30 (F) and Het-1A (G) cells treated with 0.8 μM Z4-6 and Z4-6-Dox, and 4 μM Dox. The asterisks indicate significance, ∗∗∗P < 0.001, ns indicates no significance. (H) Calcein-AM/PI imaging of KYSE30 and Het-1A cells treated with 0.8 μM Z4-6 and Z4-6-Dox, and 4 μM Dox. The green indicates Calcein-AM-stained live cells, and the red indicates PI-stained dead cells. (I) The percentage of dead cells was calculated. Untreated cells are the controls. The asterisks indicate significance, ∗∗P < 0.01, ∗∗∗P < 0.001.

To evaluate the in vitro targeting ability of Z4-6-Dox, cell internalization experiments were conducted using confocal imaging. After treatment with Z4-6-Dox, obvious Dox fluorescence was observed in the target KYSE30 cells, whereas no significant Dox fluorescence was observed in the control Het-1A cells. In contrast, free Dox was found to non-selectively enter into both target and control cells, indicating that Z4-6-Dox can be selectively internalized into the target cells (Fig. 6D and E). Subsequently, CCK-8 assays were used to evaluate the selective in vitro cytotoxicity of Z4-6-Dox. As shown in Fig. 6F and G, both Z4-6-Dox and free Dox showed comparable cytotoxicity to the target KYSE30 cells; however, Z4-6-Dox showed reduced cytotoxicity towards the control Het-1A cells compared to free Dox. These results indicate that Z4-6-Dox can selectively kill target KYSE30 cells. Furthermore, calcein-AM/PI staining was applied to further verify the selective in vitro cytotoxicity of Z4-6-Dox. As shown in Fig. 6H and I, treatment with Z4-6-Dox resulted in a significantly higher number of PI-stained KYSE30 cells compared to PI-stained Het-1A cells. In contrast, treatment with free Dox resulted in similar levels of PI-stained cells in both KYSE30 and Het-1A cells, suggesting that Z4-6-Dox exhibits selective in vitro cytotoxicity to target cells. Based on the above results, we believe that Z4-6-Dox can target to and selectively kill ESCC KYSE30 target cells.

3.7. Z4-6-Cy5 probe enables in vivo fluorescence imaging of tumors

To evaluate the in vivo targeting ability of the Z4-6 aptamer, the Cy5 fluorescent moiety was conjugated with Z4-6, resulting in the formation of the Z4-6-Cy5 fluorescent probe. The Random DNA sequence labeled with the Cy5 fluorescent moiety served as the Random-Cy5 control group. These probes were injected into KYSE30 tumor-bearing mice through the tail vein, and the distribution of the fluorescent probes in mice were detected using an in vivo fluorescence imaging system. The results of time-lapse fluorescence imaging showed that Cy5 fluorescence could be observed at tumor sites from mouse mode treated with the Z4-6-Cy5 probe (Fig. 7A), whereas no fluorescence was observed at tumor sites in mice treated with the Random-Cy5 probe (Fig. 7C). Fluorescence imaging of excised tissues corroborated that the Z4-6-Cy5 probe successfully targeted tumor sites (Fig. 7B), unlike the Random-Cy5 control probe (Fig. 7D). The quantitative analysis of fluorescence intensity at tumor sites in mice, as well as in excised tumors, provided further validation for these findings (Fig. 7E and F). Additionally, the probes exhibited significant accumulation in the kidney and liver, suggesting these organs as primary sites of probe metabolism. These findings collectively demonstrate that the Z4-6 aptamer effectively targets tumors in vivo and that the Z4-6-Cy5 probe holds promise for in vivo fluorescence imaging of ESCC. While the Z4-6-Cy5 probe demonstrates rapid accumulation at tumor sites, it undergoes significant degradation after 60 min, potentially limiting its in vivo applications. Future studies could explore chemical modifications, such as phosphorothioate, 2′-O-methyl, and locked nucleic acid modifications, to enhance the biostability of Z4-6 in vivo.

Fig. 7.

Fig. 7

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In vivo fluorescence imaging with the Z4-6-Cy5 probe. Time-lapse fluorescence imaging of mice bearing KYSE30 tumors after injected with the Z4-6-Cy5 (A) and Random-Cy5 (C) probes. Fluorescence images of excised heart (1), liver (2), spleen (3), lung (4), kidney (5), tumor (6) tissues from mouse model injected with the Z4-6-Cy5 (B) and Random-Cy5 (D) probes. (E) The quantitative analysis of fluorescence intensity at tumor sites in mice treated with the Z4-6-Cy5 and Random-Cy5 probes. (F) The quantitative analysis of fluorescence intensity in excised tumors and major organs.

3.8. Z4-6-Dox inhibits tumor cell growth in vivo with reduced cardiotoxicity

To evaluate the in vivo antitumor effect of Z4-6-Dox, the xenograft mouse model bearing KYSE30 tumors was constructed and subsequently treated with Z4-6-Dox (Fig. 8A). Z4-6-Dox was injected into the mice through tail vein every other day for a total of five doses, and the mice were sacrificed on day 11 for further analysis (Fig. 8B). As shown in Fig. 8C and D, the tumor volume and weight in mice treated with Z4-6-Dox and free Dox were significantly less than that in mice treated with PBS and Z4-6, indicating that Z4-6-Dox effectively inhibits tumor cell growth in vivo. However, no additional inhibitory effect of Z4-6-Dox on tumors was observed when compared to free Dox. We speculate that the in vivo instability of Z4-6 contributes to this outcome. Although Z4-6 demonstrates tumor-targeting capabilities in vivo, it undergoes rapid degradation within 1 h (Fig. 7A), which likely diminishes its targeting efficacy and antitumor potential. The serum stability of Z4-6-Dox was further evaluated in FBS by agarose gel electrophoresis. The results showed significant degradation of Z4-6-Dox in 100% FBS within 4 h (Fig. S11A and B), suggesting instability of Z4-6-Dox in vivo. Future efforts should focus on enhancing the stability of Z4-6, and the improvements of in-vivo antitumor efficacy of Z4-6-Dox are expected after stability optimization. To evaluate the effects of chemical modifications on Z4-6 stability, Z4-6 was subjected to phosphorothioate modification at all the sites. As shown in Fig. S11C and D, the modified Z4-6-PS and Z4-6-PS-Dox can even exist in 100% FBS for 24 h, and are more stable than unmodified Z4-6 (Fig. S11A and B), suggesting that phosphorothioate modification can significantly enhance Z4-6 stability, which preliminary validating the feasibility of stability optimization. Furthermore, the body weight of the mice was recorded during drug treatments (Fig. 8E), the results showed that the various drug treatments did not significantly impact the body weight of the mice. Additionally, the plasma pharmacokinetics of Z4-6-Dox in healthy nude mice was evaluated. As shown in Fig. S12, after administration of Z4-6-Dox, the area under the plasma concentration vs. time curve from 0 to 24 h (AUC0-24) of Dox in plasma is 1.2-times higher than that of free Dox (mean = 113.3 vs. 88.9 μg h/mL), indicating that Z4-6-Dox slightly increase the body longevity of Dox.

Fig. 8.

Fig. 8

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In vivo antitumor effects of Z4-6-Dox. (A) Schematic illustration of construction of KYSE30 tumor mouse model and drug treatment. (B) Timeline of drug administration. (C) Tumor volume records of the mice treated with Z4-6, Dox, and Z4-6-Dox. The Dox dosage was fixed at 2 mg/kg, and the molar ratio of Z4-6 to Dox is 1:5. The asterisk indicates significance, ∗P < 0.05. (D) Tumor weight of the mice on day 11. The asterisk indicates significance, ∗P < 0.05. (E) Body weight records of the mice with drug treatments. (F) HE staining of heart tissues with various treatments. Black arrow indicates myocardial abnormalities. Red arrow indicates myocardial interstitial dilation and congestion. (G) TUNEL immunostaining of heart tissues with various treatments. (H) Masson staining of heart tissues with various treatments. Black arrow indicates myocardial fibrosis.

In clinical cancer therapy, Dox is known to induce significant adverse effects, particularly cardiotoxicity, due to its lack of targeting capacity [41,42]. To evaluate whether Z4-6-Dox could alleviate the cardiotoxicity caused by Dox, HE staining was used to detect the morphological alterations in myocardial tissue. As shown in Fig. 8F, myocardial abnormalities, including myocardial interstitial dilation and congestion, could be observed in the free Dox group, but could not be found in the Z4-6-Dox group. Furthermore, TUNEL staining was used to detect the apoptosis of cardiomyocytes. As shown in Fig. 8G, an increase in apoptosis of cardiomyocytes was observed in the free Dox group, while reduced apoptosis of cardiomyocytes was noted in both the control and Z4-6-Dox groups. Masson staining, a widely used method to evaluate myocardial fibrosis [43], revealed the presence of myocardial fibrosis in the free Dox group, whereas no obvious fibrotic changes were observed in the control and Z4-6-Dox groups (Fig. 8H). These findings demonstrate that Z4-6-Dox has the potential to alleviate the cardiotoxicity of Dox in antitumor therapy. We speculate that the loading of Dox into Z4-6 may alter the in vivo distribution of Dox, as Z4-6 tends to accumulate in the kidneys and liver. This accumulation could potentially result in a decreased distribution of Dox in the heart, thereby reducing cardiotoxicity. To evaluate the toxicity of Z4-6-Dox to the liver and kidneys, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Cre) were measured by ELISA. As shown in Fig. S13, the levels of ALT, AST, BUN, and Cre in the treated mice did not alter significantly compared to the control group, indicating that Z4-6-Dox does not impart substantial toxicity to the liver and kidneys. HE staining of liver, spleen, lung, and kidney tissues further confirmed low cytotoxicity of Z4-6-Dox to these organs (Fig. S14). Collectively, Z4-6-Dox demonstrates the ability to inhibit tumor cell growth in vivo while minimizing cardiotoxicity. It is worth noting that the unmodified Z4-6 aptamer limited the antitumor efficacy of Z4-6-Dox due to its in vivo instability, improved antitumor effects are probably achieved after stability optimization and are helpful for therapeutic translation of Z4-6-Dox.

4. Conclusion

In summary, we have developed several novel aptamers using ESCC KYSE30 cells for positive selection and esophageal epithelial Het-1A cells for negative selection through Cell-SELEX. The selected aptamers can specifically bind to target KYSE30 cells, but not to control Het-1A cells. The dissociation constants of these aptamers to target cells are in the low nanomolar range, suggesting their high affinity for target cell binding. The results of binding assays to various cell types showed that these aptamers can bind to multiple cancer cells, including those of ESCC, gastric cancer, and liver cancer, highlighting their potential in cancer diagnosis and therapy. Meanwhile, a truncated aptamer, Z4-6, with comparable binding affinity was obtained through sequence optimization. With Z2 and Z4-6 aptamers as recognition elements, Cy5-labeled fluorescent aptamer probes are capable of specific imaging of ESCC tissues. Moreover, myosin 1B was identified as the molecular target of Z4-6, indicating its potential as a tumor biomarker and a therapeutic target for the diagnosis and targeted therapy of ESCC. Z4-6 is further used to construct Z4-6-Dox by noncovalent loading with the chemotherapeutic drug Dox for targeted therapy of ESCC. The results show that Z4-6-Dox can selectively kill target KYSE30 cells in vitro. Importantly, Z4-6 demonstrates the ability to localize to tumor sites and inhibit tumor cell growth in vivo while exhibiting reduced cardiotoxicity. Due to in vivo instability of unmodified Z4-6, further improvements of antitumor efficacy of Z4-6-Dox are expected after stability optimization. This work provides valuable molecular recognition tools and identifies a promising molecular target for the precise diagnosis and targeted therapy of ESCC.

CRediT authorship contribution statement

Zhaoting Wang: Data curation, Validation, Writing – original draft. Xiaoxiong Xiao: Funding acquisition, Methodology, Writing – review & editing. Tianlu Zhang: Data curation, Validation. Xiao Li: Formal analysis, Methodology. Mengmeng Ji: Formal analysis, Methodology. Yongqi Qian: Formal analysis, Software. Xue Bai: Methodology, Software. Xin Li: Software, Visualization. Jing Lu: Methodology, Visualization. Jinlu Tang: Software, Visualization. Kangdong Liu: Resources, Supervision, Writing – review & editing. Zhaohui Li: Methodology, Supervision, Writing – review & editing. Baoyin Yuan: Conceptualization, Funding acquisition, Supervision, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript.

Acknowledgment

This work was supported by the National Natural Science Foundation of China (No. 22007084), the China Postdoctoral Science Foundation (No. 2023M733215), the Undergraduate Innovation Training Program of Zhengzhou University 2025 (202510459207), the Natural Science Foundation General Program of Hunan Province (No.2022JJ40830), the Natural Science Foundation General Program of Changsha City (No. kq2014290), the National Multidisciplinary Cooperative Diagnosis and Treatment Capacity Building Project for Major Diseases (Lung Cancer, No. z027002).

Footnotes

Appendix A

Supplementary data to this article can be found athttps://doi.org/10.1016/j.mtbio.2026.102867.

Contributor Information

Zhaohui Li, Email: zhaohui.li@zzu.edu.cn.

Baoyin Yuan, Email: yuanbaoyin@zzu.edu.cn.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

The Supporting Information is available free of charge at publications website.

Multimedia component 1
mmc1.doc (5.3MB, doc)

Data availability

Data will be made available on request.

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Supplementary Materials

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Data Availability Statement

Data will be made available on request.

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